We plan to undertake a microcalorimetric investigation of the energetics of protein self-assembling processes. We will focus on two types of reactions: 1) Structural isomerizations of the urea, guanidine HCl and thermally unfolded proteins. 2) Metal activation process of alkaline phosphatase from E. Coli. In the study of protein chain folding several interesting concepts have evolved, and each awaits experimental check: (1) The slow folding reaction, and often the major kinetic phase when chromophoric absorptions are monitored, has been suggested to be due to the cis-trans isomerization of proline residues. Isomerization of other peptide bonds are also possible. The delta H of these isomerizations are small. We plan to measure delta H of the pH-dependent configurational changes of the unfolded proteins and compare these values with that of model peptides. (2) It is generally accepted that the nucleation reaction occurs in the early stage of the chain folding. To examine the nature of these structure nuclei, whether they are helical segments or hydrophobic droplets, we will measure delta H and delta Cp of the formation and dissociation of residual structures in urea, guanidine HCl and thermally unfolded ribonuclease and cytochrome c. In E. Coli alkaline phosphatase the stoichiometry of metal bindings and the nature of the binding sites will be determined by performing the thermal titration experiment. The measurement will include binding by Zn, Co, Cu... and other metal ions in Groups IIA and B, and the transition series of the Periodic Table. Equilibrium dialysis will also be done and the Scatchard plots will be constructed to separate the tight and the weak binding sites. To examine the specificity of the metal interaction the thermal titration will be extended to phosphate and inhibitor bindings in apoprotein samples preincubated with different numbers and combinations of metal ions. The concept of the anticooperativity and half-of-the-sites reactivity will be explored. The effect of magnesium ion will also be examined.